Abstract The CUL4A ubiquitin ligase gene is frequently amplified and/or overexpressed in breast cancer, hepatocellular carcinoma, mesothelioma and many other tumor types. CUL4A is also a direct transcriptional target of Wnt signaling, which is aberrantly activated in up to 63% of all breast cancers. Recent studies identified multiple substrates and cellular pathways that are subjected to ubiquitin-proteolytic control by CUL4A and are implicated in oncogenic transformation. However, the role of CUL4A in tumorigenesis remains unclear, and this knowledge gap is largely due to the lack of in vivo tumor models in which CUL4A expression can be manipulated in a temporal and tissue-specific manner during the course of tumor development. This proposal seeks to definitively determine the pathophysiological role of CUL4A in mammary tumorigenesis, and begins to dissect the specific ubiquitin-proteolytic events that underlie tumor initiation, maintenance, and resistance to camptothecin-type chemotherapy drugs. We are uniquely positioned to perform the proposed studies, as (1) we recently generated conditional CUL4A knockout mice that will overcome the limitations of unavailable experimental models, (2) we have at our disposal a well-established breast tumor bank with over 3,000 specimens, including over 250 triple negative breast cancer (TNBC) cases (CUL4A amplification was detected in ~20% TNBCs.), and patient clinico-pathological information, and (3) we discovered a novel function of the Cockayne syndrome A (CSA) DNA repair protein in CUL4 (CUL4A and its family member CUL4B)- dependent degradation of topoisomerase 1 (TOP1), implicating CUL4 dysregulation as a mechanism for resistance of tumor patients to TOP1-directed chemotherapy (e.g. camptothecin). We plan to test the hypothesis that CUL4A dysregulation promotes tumorigenesis and confers resistance to chemotherapy by pursuing two specific aims: (1) Determine the role and mechanistic basis of CUL4A in mammary tumor development and maintenance; (2) Delineate the mechanistic role of CUL4 dysregulation in tumor resistance to topoisomerase I inhibitor chemotherapy drugs. Successful completion of the proposed studies is anticipated to definitively establish the role of CUL4A- mediated ubiquitination in the pathogenesis of breast cancer, and shed light on the resistance of certain tumors to chemotherapy by camptothecin-type drugs. The evaluation of the efficacy of CUL4 inhibition using a combination of cell culture, mouse models and human breast cancer specimens is expected to facilitate the development of new and effective therapeutic strategies against breast cancer and other tumor types with dysregulated CUL4A expression, such as those with aberrant activation of Wnt signaling.